Cytoskeletal activities mediated by actin filaments facilitate the production of spermatozoa. Actin filaments have been identified in the Sertoli cell and germ cells but since localization data is incomplete, a major objective of this proposal is to undertake a comprehensive localization of actin within Sertoli cells and germ cells. To obtain data related to the functional activity of actin filaments, the actin filament disrupting agent--cytochalasin D--will be injected intratesticularly. A dramatic effect of cytochalasin D is to disrupt actin filaments in peritubular myoid cells. It is noted that within hours of cytochalasin D injection sperm are released normally in the appropriate stage but do not move down the seminiferous tubule. We propose studies using cytochalasin D to determine the role of myoid cells in sperm transport. Cytochalasin D prevents formation of the actin filament bundles (ectoplasmic specialization) of the Sertoli cell which face the newly elongating spermatid. The consequence of disruption of the ectoplasmic specialization on germ cell orientation and indention into the Sertoli cell will be studied. Cytochalasin D also prevents formation of the actin filaments surrounding the tubulobulbar complex--a structure which is implicated in sperm release and elimination of excess spermatid cytoplasm. The consequence of disruption of tubulobulbar complexes will be studied. Another effect of cytochalasin D is to inhibit migration of spermatocytes through the Sertoli cell barrier and to disrupt ectoplasmic specialization which lines the Sertoli cell barrier. The consequence of disruption of the ectoplasmic specialization in prepubertal animals just forming a Sertoli cell barrier as well as in the maintenance of the barrier in the adult will be studied with transmission electron microscopy and also freeze-fracture to determine if actin is involved with the formation or regulation of occluding junctions. Cytochalasin D also has a rapid effect on intercellular bridges between spermatids, causing them to open. The intercellular bridges are remnants of the actin-containing contractile ring and show 5-7 nm filaments. It has been proposed that synchronization of germ cell development occurs through bridges. Collectively, the processes to be examined are fundamental to the successful maturation and transport of germ cells in all mammals including man and their selective disruption will provide valuable information on the normal mechanisms of sperm production as well as potential insights into pathologies which affect spermatogenesis by similar mechanisms.